Redefining The Role Of Myosin Essential Light Chain In Cardiac Muscle

重新定义肌球蛋白必需轻链在心肌中的作用

• 批准号: 10376748
• 负责人: Danuta Szczesna-Cordary
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-05 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376748
• 关键词: Actins; Age; Alkalies; Amino Acids; Binding; Biological Assay; Calcium; Cardiac; Cardiac Muscle Contraction; Cardiomyopathies; Cells; Complex; Consumption; Data; Disease; Echocardiography; Etiology; Event; Fluorescence; Fluorescent Probes; Frequencies; Genes; Glycine; Head; Health; Heart; Heart Diseases; Homeostasis; Human; Hypertrophic Cardiomyopathy; Hypertrophy; In Vitro; Investigation; Label; Light; Link; Lysine; Measurement; Metabolic; Mitochondria; Modeling; Modification; Molecular; Motor; Mus; Muscle; Muscle Contraction; Muscle Fibers; Mutate; Mutation; Myocardium; Myosin ATPase; Myosin Alkali Light Chains; N-terminal; Organ; Organelles; Output; Oxidative Phosphorylation; Pathologic; Peroxisome Proliferator-Activated Receptors; Phenotype; Physiological; Play; Production; Proteins; Quantum Dots; RNA Splicing; Regulation; Respiration; Respiratory Chain; Role; Skeletal Muscle; Skin; Smooth Muscle Myosins; System; Testing; Thick Filament; Transgenic Mice; Transgenic Organisms; Variant; Ventricular; Work; base; cardiac muscle disease; cell motility; hemodynamics; hexokinase; in vivo; insight; mouse model; mutant; novel; novel therapeutics; papillary muscle; respiratory; response; sex

ABSTRACT The scientific premise of this application regards the cardiac specific expression of the long ELC containing the N-terminal ELC extension (N-ELC) and its physiologically important role in regulating myosin motor function and force production in muscle. We hypothesize that cardiac N-ELC works as a molecular linker and/or energetic switch of the actin-myosin interaction by regulating the transition of myosin cross-bridges from a super- relaxed (SRX) to a disordered-relaxed (DRX) state, and the proportion of myosin heads that are available for the interaction with actin. In DRX, the myosin heads protrude into the interfilament space but are restricted from binding to actin, while in the SRX state, the heads are neatly ordered along the thick filament axis but cycle at a highly inhibited ATP turnover rate. We further hypothesize that hypertrophic cardiomyopathy mutations in myosin ELC promote the SRX-to-DRX transition and the deletion of the N-ELC counteracts this transition and stabilizes the energy conservation SRX state. We will test these hypotheses using two mouse models of hypertrophic HCM-A57G (Alanine57Glycine) and restrictive RCM-E143K (Glutamate143Lysine) cardiomyopathy. We will also use transgenic Δ43 mice expressing a 43-aa truncated ELC, and double transgenic mice: A57GxΔ43 and E143KxΔ43, expressing Δ43-ELC in the background of HCM or RCM mutations. The data will be compared to age and sex matched WT-ELC mice expressing the human ventricular ELC (MYL3 gene). SPECIFIC AIM 1: DEFINE THE ROLE OF MYOSIN ELC AND ITS N-TERMINUS (N-ELC) IN THE REGULATION OF MYOSIN MOTOR FUNCTION AND CARDIAC MUSCLE CONTRACTION. This aim will focus on the molecular triggers of ELC-induced heart remodeling studied at the level of myosin molecules, myofibers and the whole heart. The N-ELC-dependent regulation of actin-myosin interactions will be studied using: 1) mant-ATP-chase assays and the proportion of myosin heads in SRX vs. DRX states; 2) Quantum dot-labeled actin±Tm-Tn motility assays; 3) Skinned and intact muscle fibers and force-pCa as well as force and calcium transient measurements; and 4) echocardiography and invasive hemodynamics to assess contractile responses of the heart in vivo. SPECIFIC AIM 2: THE ROLE MYOSIN ELC IN ENERGETIC REMODELING OF THE HEART IN MOUSE MODELS OF CARDIOMYOPATHY. The analysis of the metabolic landscape in A57G, E143K, Δ43, A57GxΔ43 and E143KxΔ43 vs. WT hearts will be performed to identify the specific mechanisms that govern energy utilization in all ELC-mutant vs. WT mice. Mitochondrial function will be assessed by determination of mitochondrial respiration, enzymatic activity of respiratory complexes as well as ATP production/consumption. Steady-state levels of candidate proteins implicated in energy production such as Hexokinases I and II, various mitochondrial respiratory chain subunits (Complexes I to V) as well as peroxisome proliferator-activated receptor coactivator α (PGC-1α) will be assessed in all models to characterize the broader implications of cardiac muscle disease resulting from ELC-linked modifications.

摘要 本申请的科学前提是考虑含有长ELC的心脏特异性表达。 N-末端ELC延伸（N-ELC）及其在调节肌球蛋白运动功能中重要生理作用 和肌肉的力量产生。我们假设心脏N-ELC作为分子接头和/或 肌动蛋白-肌球蛋白相互作用的能量开关，通过调节肌球蛋白跨桥的过渡， 松弛（SRX）到无序松弛（DRX）状态的肌球蛋白头的比例，以及肌球蛋白头的比例。 与肌动蛋白的相互作用在DRX中，肌球蛋白头突出到肌丝间隙中，但被限制从肌丝间隙中伸出。 结合肌动蛋白，而在SRX状态，头部整齐地沿着粗丝轴排列，但在一个周期， 高度抑制ATP周转率。我们进一步假设肥厚型心肌病基因突变可能与心肌细胞的凋亡有关。 肌球蛋白ELC促进SRX到DRX的转变，N-ELC的缺失抵消了这种转变， 使节能SRX状态稳定。我们将使用两种小鼠模型来测试这些假设。 肥厚型HCM-A57 G（丙氨酸57-甘氨酸）和限制性RCM-E143 K（谷氨酸143-赖氨酸） 心肌病我们还将使用表达43-aa截短ELC的转基因Δ43小鼠，以及双转基因Δ43小鼠。 小鼠：A57 Gx Δ43和E143 Kx Δ43，在HCM或RCM突变背景下表达Δ43-ELC。数据 将与表达人心室ELC（MYL 3基因）的年龄和性别匹配的WT-ELC小鼠进行比较。 具体目的1：确定肌球蛋白ELC及其N-乙酰基（N-ELC）在调节 肌球蛋白运动功能和心肌收缩。这一目标将集中在分子 在肌球蛋白分子、肌纤维和整体水平研究ELC诱导的心脏重塑的触发因素 心N-ELC依赖的肌动蛋白-肌球蛋白相互作用的调节将使用：1）mant-ATP-chase 测定和SRX与DRX状态中肌球蛋白头的比例; 2）量子点标记的肌动蛋白±Tm-Tn运动性 3）去皮和完整的肌纤维和力-pCa以及力和钙瞬变测量; 和4）超声心动图和侵入性血流动力学以评估体内心脏的收缩反应。 特定目标2：肌球蛋白ELC在小鼠模型心脏血管重建中的作用 心肌病。对A57 G、E143 K、Δ43、A57 Gx Δ43和A57 Gx Δ43中的代谢景观进行了分析。 将进行E143 Kx Δ43与WT心脏的比较，以确定控制能量利用的具体机制 在所有ELC突变体与WT小鼠中。将通过测定线粒体功能来评估线粒体功能。 呼吸、呼吸复合物的酶活性以及ATP的产生/消耗。稳态 参与能量产生的候选蛋白质水平，例如己糖激酶I和II，各种线粒体 呼吸链亚单位（复合物I至V）以及过氧化物酶体增殖物激活受体辅激活因子α 将在所有模型中评估PGC-1α，以表征心肌疾病的更广泛意义 由ELC连接的修饰产生。